I am trying to see colocalization of two proteins in confocal where one is a fusion GFP transfected and the other stained for the endogenous and detected using Alexa 568. I am no able to see cells both with GFP and red marker. But as such independently I can see lot of red cells. Is transfection masking the effect of endogenous in microscopy? Should I use Ab staining or both as I have two different host Abs.
Which proteins are you investigating? I would definitely start with looking at endogenous proteins with your two antibodies. Also, you could try titrating down the amount of GFP construct to see if that will increase the endogenous one. Also, if you transfect the empty GFP vector do you see your other protein of interest? Answering this question will let you know if the reduction in your other protein upon transfection is specific for your GFP-tagged protein or not. Best of luck!
Hi Darla,
I am looking at a glutamate transporter in GFP fusion and endogenous dendritic marker. Lesser GFP transfection reduces my efficiency of transfection. Yeah I do see endogenous expression of the marker in both empty GFP and fusion but problem is I am not getting colocalization. I am seeing independent cells. I suspect a masking effect of GFP. I expect a coloc. I tried Ab staining today, it looks fine but cell density has to be really reduced. I am working with glioma cells so I need better space n low cell no for them to spread enough.
It is possible the addition of GFP to your protein prevents your antibody from binding. Is your antibody directed toward one of the protein termini where the GFP tag is? It is possible that overexpression is masking the endogenous protein. To check this, I would run a Western blot on cell lysates to see if you are getting both forms (the GFP version should run about 25 kDa higher on the gel). Good luck!
- Badges
- Science method